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1.
Cell Rep Methods ; 3(4): 100443, 2023 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-37159665

RESUMO

Unintegrated HIV DNA represents between 20% and 35% of the total viral DNA in infected patients. Only the linear forms (unintegrated linear DNAs [ULDs]) can be substrates for integration and for the completion of a full viral cycle. In quiescent cells, these ULDs may be responsible for pre-integrative latency. However, their detection remains difficult due to the lack of specificity and sensitivity of existing techniques. We developed an ultra-sensitive, specific, and high-throughput technology for ULD quantification called DUSQ (DNA ultra-sensitive quantification) combining linker-mediated PCR and next-generation sequencing (NGS) using molecular barcodes. Studying cells with different activity levels, we determined that the ULD half-life goes up to 11 days in resting CD4+ T cells. Finally, we were able to quantify ULDs in samples from patients infected with HIV-1, providing a proof of concept for the use of DUSQ in vivo to track pre-integrative latency. DUSQ can be adapted to the detection of other rare DNA molecules.


Assuntos
Soropositividade para HIV , HIV-1 , Humanos , DNA Viral/genética , Tecnologia , Divisão Celular , HIV-1/genética
2.
Microbiol Spectr ; 10(4): e0078422, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35770985

RESUMO

In HIV infection, viral rebound after treatment discontinuation is considered to originate predominantly from viral genomes integrated in resting CD4+ T lymphocytes. Replication-competent proviral genomes represent a minority of the total HIV DNA. While the quantification of the HIV reservoir has been extensively studied, the diversity of genomes that compose the reservoir was less explored. Here, we measured the genotypic and phenotypic diversity in eight patients with different treatment histories. Between 4 and 14 (mean, 8) individual viral isolates per patient were obtained using a virus outgrowth assay, and their near-full-length genomes were sequenced. The mean pairwise distance (MPD) observed in different patients correlated with the time before undetectable viremia was achieved (r = 0.864, P = 0.0194), suggesting that the complexity of the replication-competent reservoir mirrors that present at treatment initiation. No correlation was instead observed between MPD and the duration of successful treatment (mean, 8 years; range, 2 to 21 years). For 5 of the 8 patients, genotypically identical viral isolates were observed in independent wells, suggesting clonal expansion of infected cells. Identical viruses represented between 25 and 60% of the isolates (mean, 48%). The proportion of identical viral isolates correlated with the duration of treatment (r = 0.822, P = 0.0190), suggesting progressive clonal expansion of infected cells during ART. A broader range of infectivity was also observed among isolates from patients with delayed viremia control (r = 0.79, P = 0.025). This work unveiled differences in the genotypic and phenotypic features of the replication-competent reservoir from treated patients and suggests that delaying treatment results in increased diversity of the reservoir. IMPORTANCE In HIV-infected and effectively treated individuals, integrated proviral genomes may persist for decades. The vast majority of the genomes, however, are defective, and only the replication-competent fraction represents a threat of viral reemergence. The quantification of the reservoir has been thoroughly explored, while the diversity of the genomes has been insufficiently studied. Its characterization, however, is relevant for the design of strategies aiming the reduction of the reservoir. Here, we explored the replication-competent near-full-length HIV genomes of eight patients who experienced differences in the delay before viremia control and in treatment duration. We found that delayed effective treatment was associated with increased genetic diversity of the reservoir. The duration of treatment did not impact the diversity but was associated with higher frequency of clonally expanded sequences. Thus, early treatment initiation has the double advantage of reducing both the size and the diversity of the reservoir.


Assuntos
Antirretrovirais , Infecções por HIV , Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos , Genoma Viral , Infecções por HIV/tratamento farmacológico , Humanos , Provírus/genética , Carga Viral , Viremia/tratamento farmacológico , Latência Viral
3.
J Virol ; 95(5)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33298540

RESUMO

Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that reduces HIV-1 infectivity by an incompletely understood mechanism. We show here that viruses differing only in the envelope glycoprotein (Env) expressed on their surface have different sensitivities to IFITM3. Measurements of the sensitivity of viruses to neutralizing antibodies showed that IFITM3 increased the sensitivity of IFITM3-sensitive viruses to PG16, which targets the V1V2 loop, suggesting that IFITM3 promotes exposure of the PG16 epitope of IFITM3-sensitive viruses. Exchanges of V1V2 loops between the Env proteins of sensitive and resistant viruses revealed that V1V2 and V3 act together to modulate viral sensitivity to IFITM3. Co-immunoprecipitation experiments showed that IFITM3 interacted with both the precursor (gp160) and cleaved (gp120) forms of Env from IFITM3-sensitive viruses, but only with the precursor (gp160) form of Env from IFITM3-resistant viruses. This finding suggests that the interaction between the Env of resistant viruses and IFITM3 was inhibited once Env had been processed in the Golgi apparatus. This hypothesis was supported by immunofluorescence experiments, which showed a strong colocalization of IFITM3 with the Env of sensitive viruses, but only weak colocalization with the Env of resistant viruses on the plasma membrane of virus-producing cells. Together, these results indicate that IFITM3 interacts with Env, inducing conformational changes that may decrease viral infectivity. This antiviral action is, nevertheless, modulated by the nature of the Env, in particular its V1V2 and V3 loops, which after maturation may be able to escape this interaction.IMPORTANCE Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that reduces HIV-1 infectivity by an incompletely understood mechanism. This study aimed to elucidate the role of the HIV-1 envelope glycoprotein (Env) in determining viral susceptibility to IFITM3. We found that viruses differing only in Env expressed on their surface had different sensitivities to IFITM3. By comparing the Env proteins of viruses that were highly sensitive or resistant to IFITM3, we obtained new insight in the mechanisms by which HIV-1 escapes this protein. We showed that IFITM3 interacts with the Env protein of sensitive viruses in virion-producing cells, inducing conformational changes that may decrease viral infectivity. However, this antiviral action is modulated by the nature of Env, particularly the V1V2 and V3 loops, which may be able to escape this interaction after processing in the Golgi.

4.
Sci Rep ; 10(1): 16744, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028961

RESUMO

The diversity of the HIV-1 envelope glycoproteins (Env) is largely a consequence of the pressure exerted by the adaptive immune response to infection. While it was generally assumed that the neutralizing antibody (NAb) response depended mainly on the infected individual, the concept that virus-related factors could be important in inducing this response has recently emerged. Here, we analyzed the influence of the infecting viral strain in shaping NAb responses in four HIV-1 infected subjects belonging to a transmission chain. We also explored the impact of NAb responses on the functional evolution of the viral quasispecies. The four patients developed a strong autologous neutralizing antibody response that drove viral escape and coincided with a parallel evolution of their infecting quasispecies towards increasing infectious properties, increasing susceptibility to T20 and increasing resistance to both CD4 analogs and V3 loop-directed NAbs. This evolution was associated with identical Env sequence changes at several positions in the V3 loop, the fusion peptide and the HR2 domain of gp41. The common evolutionary pattern of Env in different hosts suggests that the capacity of a given Env to adapt to changing environments may be restricted by functional constraints that limit its evolutionary landscape.


Assuntos
Evolução Molecular , Infecções por HIV/virologia , HIV-1/metabolismo , Proteínas do Envelope Viral/metabolismo , Humanos , Masculino , Monócitos/metabolismo , Monócitos/virologia
5.
AIDS ; 34(15): 2187-2200, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32932339

RESUMO

OBJECTIVE: HIV-1 transmission leads to a genetic bottleneck, with one or a few variants of the donor quasispecies establishing an infection in the new host. We aimed to characterize this bottleneck in more detail, by comparing the properties of HIV envelope glycoproteins from acute and chronic infections within the particular context of a male-to-male transmission cluster. DESIGN: We compared the genotypic and phenotypic properties of envelope glycoproteins from viral variants derived from five study participants from the same transmission cluster. METHODS: We used single-genome amplification to generate a collection of full-length env sequences. We then constructed pseudotyped viruses expressing selected Env variants from the quasispecies infecting each study participant and compared their infectivities and sensitivities to various entry inhibitors. RESULTS: The genotypic analyses confirmed the genetic bottleneck expected after HIV transmission, with a limited number of variants identified in four study participants during acute infection. However, the transmitted sequences harbored no evident common signature and belonged to various genetic lineages. The phenotypic analyses revealed no difference in infectivity, susceptibility to the CCR5 antagonist maraviroc, the fusion inhibitor enfurvitide or type-I interferon between viruses from participants with acute and chronic infections. The key property distinguishing transmitted viruses was a higher resistance to soluble CD4, correlated with greater sensitivity to occupation of the CD4 receptor by the anti-CD4 antibodies LM52 and SK3. CONCLUSION: These results suggest that envelope glycoproteins from transmitted/founder viruses bind CD4 less efficiently than those of viruses from chronic infections.


Assuntos
Linfócitos T CD4-Positivos , Glicoproteínas , Infecções por HIV , HIV-1 , Homossexualidade Masculina , Proteínas do Envelope Viral , Linfócitos T CD4-Positivos/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Masculino , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
6.
J Virol ; 92(4)2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29187544

RESUMO

HIV-infected subjects under antiretroviral treatment (ART) harbor a persistent viral reservoir in resting CD4+ T cells, which accounts for the resurgence of HIV replication after ART interruption. A large majority of HIV reservoir genomes are genetically defective, but even among intact proviruses few seem able to generate infectious virus. To understand this phenomenon, we examined the function and expression of HIV envelope glycoproteins reactivated from the reservoir of four HIV-infected subjects under suppressive ART. We studied full-length genetically intact env sequences from both replicative viruses and cell-associated mRNAs. We found that these Env proteins varied extensively in fusogenicity and infectivity, with strongest functional defects found in Envs from cell-associated mRNAs. Env functional impairments were essentially explained by defects in Env protein expression. Our results support the idea that defects in HIV Env expression, preventing cytopathic or immune HIV clearance, contribute to the persistence of the HIV T-cell reservoir in vivoIMPORTANCE In most individuals, evolution of HIV infection is efficiently controlled on the long-term by combination antiviral therapies. These treatments, however, fail to eradicate HIV from the infected subjects, a failure that results both in resurgence of virus replication and in resumption of HIV pathogenicity when the treatment is stopped. HIV resurgence, in these instances, is widely assumed to emerge from a reservoir of silent virus integrated in the genomes of a small number of T lymphocytes. The silent HIV reservoir is mostly composed of heavily deleted or mutated HIV DNA. Moreover, among the seemingly intact remaining HIV, only very few are actually able to efficiently propagate in tissue culture. In this study, we find that intact HIV in the reservoir often carry strong defects in their capacity to promote fusion to neighboring cells and infection of target cells, a defect related to the function and expression of the HIV envelope glycoprotein. Impaired envelope glycoprotein expression and function could explain why cells harboring these viruses tend to remain undetected and unharmed in the reservoir.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/genética , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Células Cultivadas , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Provírus , Latência Viral
7.
Proc Natl Acad Sci U S A ; 110(44): 17957-62, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24133137

RESUMO

Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during differentiation of the myelomonoblast PLB-985 cell line into "neutrophil-like" cells. Functional assays indicated that human leukocyte antigen G, the preferred ligand of ILT4, inhibited the phagocytic function of neutrophils. ILT4 engagement also impaired reactive oxygen species production induced through CD32a and both receptors were found colocalized into neutrophil lipid rafts. Moreover, neutrophil degranulation induced through inflammatory stimuli increased ILT4 expression as a result of the rapid translocation of an intracellular pool to the cell surface. Consequently to this ILT4 up-regulation, the human leukocyte antigen G-mediated inhibition of neutrophil phagocytic function was enhanced. Finally, we found that ILT4 up-regulation induced on healthy donor neutrophils following stimulation was impaired in presence of plasma from patients with sepsis. Similarly, ILT4 up-regulation was inhibited in neutrophils from septic patients. Altogether, our results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders.


Assuntos
Exocitose/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Neutrófilos/imunologia , Receptores Imunológicos/imunologia , Explosão Respiratória/imunologia , Apoptose , Citometria de Fluxo , Humanos , Microscopia Confocal , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , Sepse/imunologia , Sepse/metabolismo
8.
Retrovirology ; 10: 50, 2013 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-23647667

RESUMO

BACKGROUND: HIV-2, which was transmitted to humans from a distant primate species (sooty mangabey), differs remarkably from HIV-1 in its infectivity, transmissibility and pathogenicity. We have tested the possibility that a greater susceptibility of HIV-2 capsid (CA) to the human restriction factor TRIM5α (hTRIM5α) could contribute to these differences. RESULTS: We constructed recombinant clones expressing CA from a variety of HIV-2 viruses in the context of HIV-1 NL4-3-luciferase. CA sequences were amplified from the plasma of HIV-2 infected patients, including 8 subtype A and 7 subtype B viruses. CA from 6 non-epidemic HIV-2 subtypes, 3 HIV-2 CRF01_AB recombinants and 4 SIVsmm viruses were also tested. Susceptibility to hTRIM5α was measured by comparing single-cycle infectivity in human target cells expressing hTRIM5α to that measured in cells in which hTRIM5α activity was inhibited by overexpression of hTRIM5γ.The insertion of HIV-2 CA sequences in the context of HIV-1 did not affect expression and maturation of the HIV-2 CA protein. The level of susceptibility hTRIM5α expressed by viruses carrying HIV-2 CA sequences was up to 9-fold higher than that of HIV-1 NL4-3 and markedly higher than a panel of primary HIV-1 CA sequences. This phenotype was found both for viruses carrying CA from primary HIV-2 sequences and viruses carrying CA from laboratory-adapted HIV-2 clones. High hTRIM5α susceptibility was found in all HIV-2 subtypes. In this series of viruses, susceptibility to hTRIM5α was not significantly affected by the presence of a proline at position 119 or by the number of prolines at positions 119, 159 or 178 in HIV-2 CA. No significant correlation was found between HIV-2 viremia and sensitivity to hTRIM5α. CONCLUSIONS: HIV-2 capsid sequences expressed high levels of susceptibility to hTRIM5α. This property, common to all HIV-2 sequences tested, may contribute in part to the lower replication and pathogenicity of this virus in humans.


Assuntos
Proteínas do Capsídeo/imunologia , Proteínas de Transporte/imunologia , HIV-2/imunologia , Fatores de Restrição Antivirais , Humanos , Estabilidade Proteica , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
9.
Eur J Immunol ; 42(3): 700-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22144141

RESUMO

HLA-G is a non-classical HLA class I molecule with tolerogenic properties and restricted tissue distribution. The expression of HLA-G can be induced by tumors thus providing an efficient way to escape the anti-tumoral immune response. Although lipid rafts regulate diverse immunological mechanisms their relationship with HLA-G remains controversial. Our results show that HLA-G-mediated inhibition of both the interaction between NK and tumor cells, and of intracellular calcium flux in NK cells conjugated to their target cells were independent of lipid raft integrity. In addition, cytotoxicity assays indicated that HLA-G continued to efficiently inhibit NK-cell cytolytic function in several different tumor cells independently of lipid raft integrity. Confocal microscopy with 3D reconstruction combined with biochemical analysis showed that HLA-G was mainly localized outside the lipid rafts of tumor cells after cross-linking with specific antibody and remained excluded from lipid rafts during interaction with the ILT2 inhibitory receptor of NK cells. This study indicates that the inhibitory function of HLA-G is uncoupled from lipid raft organization, further distinguishing HLA-G from classical HLA molecules and providing novel information in the understanding of tumor immune escape mechanism mediated through HLA-G.


Assuntos
Antígenos HLA-G/imunologia , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Microdomínios da Membrana/imunologia , Antígenos CD/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Antígenos HLA-G/genética , Humanos , Células Matadoras Naturais/ultraestrutura , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Microdomínios da Membrana/ultraestrutura , Microscopia Confocal , Receptores Imunológicos/imunologia , Evasão Tumoral/imunologia , beta-Ciclodextrinas/farmacologia
10.
J Virol ; 85(22): 11846-54, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21917976

RESUMO

Although laboratory-adapted HIV-1 strains are largely resistant to the human restriction factor TRIM5α (hTRIM5α), we have recently shown that some viruses carrying capsid (CA) sequences from clinical isolates can be more sensitive to this restriction factor. In this study we evaluated the contribution to this phenotype of CA mutations known to be associated with escape from cytotoxic T lymphocyte (CTL) responses. Recombinant viruses carrying HIV-1 CA sequences from NL4-3 and three different clinical isolates were prepared, along with variants in which mutations associated with CTL resistance were modified by site-directed mutagenesis, and the infectivities of these viruses in target cells expressing hTRIM5α and cells in which TRIM5α activity had been inhibited by overexpression of TRIM5γ were compared. For both hTRIM5α-sensitive viruses studied, CTL-associated mutations were found to be responsible for this phenotype. Both CTL resistance mutations occurring within HLA-restricted CA epitopes and compensatory mutations occurring outside CTL epitopes influenced hTRIM5α sensitivity, and mutations associated with CTL resistance selected in prior hosts can contribute to this effect. The impact of CTL resistance mutations on hTRIM5α sensitivity was context dependent, because mutations shown to be responsible for the TRIM5α-sensitive phenotype in viruses from one patient could have little or no impact on this parameter when introduced into another virus. No fixed relationship between changes in hTRIM5α sensitivity and infectivity was discernible in our studies. Taken together, these findings suggest that CTL mutations may influence HIV-1 replication by modifying both viral infectivity and sensitivity to TRIM5α.


Assuntos
Proteínas de Transporte/imunologia , HIV-1/imunologia , HIV-1/patogenicidade , Mutação de Sentido Incorreto , Linfócitos T Citotóxicos/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Fatores de Restrição Antivirais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas de Transporte/metabolismo , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos , Mutagênese Sítio-Dirigida , Recombinação Genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
11.
J Virol ; 85(15): 7828-35, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21632761

RESUMO

TRIM5α is a restriction factor that can block an early step in the retroviral life cycle by recognizing and causing the disassembly of incoming viral capsids, thereby preventing the completion of reverse transcription. Numerous other isoforms of human TRIM5 exist, and isoforms lacking a C-terminal SPRY domain can inhibit the activity of TRIM5α. Thus, TRIM5α activity in a given cell type could be dependent on the relative proportions of TRIM5 isoforms expressed, but little information concerning the relative expression of TRIM5 isoforms in human cells is available. In this study, we demonstrate that mRNAs coding for TRIM5α represent only 50% of total TRIM5 transcripts in human cell lines, CD4(+) T cells, and macrophages. Transcripts coding for, in order of abundance, TRIM5ι (TRIM5-iota), a previously uncharacterized isoform, TRIM5γ, TRIM5δ, and TRIM5κ are also present. Like TRIM5γ and TRIM5δ, TRIM5ι and TRIM5κ do not inhibit HIV-1 replication, but both have dominant-negative activity against TRIM5α. Specific knockdown of TRIM5ι increases TRIM5α activity in human U373-X4 cells, indicating that physiological levels of expression of truncated TRIM5 isoforms in human cells can reduce the activity of TRIM5α.


Assuntos
Processamento Alternativo , Proteínas de Transporte/fisiologia , Isoformas de Proteínas/fisiologia , Fatores de Restrição Antivirais , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Reação em Cadeia da Polimerase , Isoformas de Proteínas/química , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases
12.
J Virol ; 84(21): 11010-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20702630

RESUMO

HIV-1 infectivity is strongly restricted by TRIM5α from certain primate species but has been described as being only marginally susceptible to human TRIM5α. In this study, we evaluated the effects of the modulation of human TRIM5α activity (pretreatment of target cells with alpha interferon, expression of a pre-miRNA targeting TRIM5α, and/or overexpression of TRIM5γ), the inhibition of cyclophilin A (CypA)-CA interactions, and the expression of different allelic variants of human TRIM5α on the infectivity of a series of recombinant viruses carrying different patient-derived Gag-protease sequences. We show that HIV-1 displays virus-specific differences in its sensitivity to human TRIM5α and in its sensitivity to different TRIM5α alleles. The effect of inhibiting CypA-CA interactions is also strain specific, and blocking these interactions can either inhibit or improve viral infectivity, depending on the isolate studied. The inhibition of CypA-CA interactions also modulates viral sensitivity to human TRIM5α. In the absence of CypA-CA interactions, most viruses displayed increased sensitivity to the inhibitory effects of TRIM5α on viral replication, but one isolate showed a paradoxical decrease in sensitivity to TRIM5α. Taken together, these findings support a model in which three interlinked factors--capsid sequence, CypA levels, and TRIM5α--interact to determine capsid stability and therefore viral infectivity.


Assuntos
Alelos , Capsídeo/metabolismo , Proteínas de Transporte/fisiologia , Ciclofilina A/metabolismo , HIV-1/patogenicidade , Fatores de Restrição Antivirais , Sequência de Bases , Proteínas de Transporte/genética , Ciclofilina A/análise , Produtos do Gene gag/genética , Humanos , Especificidade da Espécie , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Replicação Viral
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